Extracts of Echinodorus grandiflorus obtained from
dried leaves by three different techniques were evaluated by bacterial lysogenic
induction assay (Inductest) in relation to their genotoxic properties.
Before being added to test cultures, extracts were sterilized either by steam
sterilization or ultraviolet light. Only the extracts prepared by infusion and
steam sterilized have shown genotoxic activity. The phytochemical analysis
revealed the presence of the flavonoids isovitexin, isoorientin, swertisin and
swertiajaponin, isolated from a genotoxic fraction. They were assayed separately
and tested negative in the Inductest protocol. The development of browning color
and sweet smell in extracts submitted to heat, prompted further chemical
analysis in search for Maillard's reaction precursors. Several aminoacids
and reducing sugars were cast in the extract. The presence of characteristic
Maillard's melanoidins products was determined by spectrophotometry in the
visible region and the inhibition of this reaction was observed when its
characteristic inhibitor, sodium bisulfite, was added prior to heating.
Remarkably, this is the first paper reporting on the appearance of such
compounds in a phytomedicine preparation under a current phytopharmaceutical
procedure. The genotoxic activity of such heat-prepared infusions imply in some
risk of developing degenerative diseases for patients in long-term, uncontrolled
use of such phytomedicines.

Echinodorus grandiflorus species belong to a worldwide
spread family Alismataceae. It is popularly known as chapéu de
couro in Brazil, with a strong diuretic effect attributed to infusions
made from its leaves. Industrially, it is one of the main ingredients for popular
soft drinks, being cultivated for this purpose in the Rio de Janeiro and Minas
Gerais regions. Antinociceptive, antiinflamatory and hypocholesterolemic activities
were also reported to occur in addition to the diuretic one (Cardoso et al. 2003). A recent in
vitro study suggests that E. grandiflorus aqueous
extracts may modulate pulmonary allergic responses (Brugiolo et al. 2011).
Exposure of renal cells to Echinodorus extracts however, have
induced genotoxic effects (Lopes et al.
2000), corroborated by results of increased mutagenic and lysogenic
induction in bacterial cells (Vidal et al.
2010).

Phytochemical evaluation of extracts made from leaves has led to the
identification of cembrane and clerodane-type diterpenoids, sesquiterpenes and
flavonoids (Schnitzler et al. 2007,
Costa et al. 1999, Tanaka et al. 1997,
Manns and Hartmann 1993).
Maillard's products, those originated from the reaction between aminoacids and
reducing sugars upon heating, are well known mutagenic compounds and their formation
in food products is kept to a minimum to prevent loss of quality and nutritional
properties (Powrie et al. 1986).
Inversely to Food Chemistry, little effort has been made in the field of medicinal
plant chemistry to identify Maillard's reaction products in phytomedicines and
popular plant preparations. The genotoxic and mutagenic effects detected so far,
deriving from exposure to such preparation, might find their cause on the presence
of such Maillard's products due to heat sterilization procedures.

In this paper we report the formation of melanoidin, a known mutagenic
compound, in infusions made of Echinodorus grandiflorus and test
the influence of other sterilization processes and pharmaceutical preparations on
the genotoxic activity of the extract.

MATERIALS AND METHODS

Plant

Echinodorus grandiflorus were kindly provided by a
local pharmaceutical industry (Laboratório Simões Ltd., Rio de
Janeiro, Brazil) after being collected (Nova Friburgo, Rio de Janeiro,
Brazil) in April 2004 and identified by Dr. Yara Lucia Oliveira de Britto
from the Rio de Janeiro Botanical Garden (Brazil), where an exsiccate
sample is deposited.

Preparation of Extracts and Solutions

Aqueous extract: 200 g dried leaves were sonicated in
1000 mL water during 10 minutes. After this procedure, the extract was filtered
in paper and the initial volume restored with distilled water. Ethanol
extract: 400 g dried leaves were immersed in 2000 mL 96° ethanol
and macerated during 24 hours. After this procedure, the extract was filtered in
paper and the initial volume restored with 96° ethanol.
Infusion: 100 mL boiling water was added to 10 g dried leaves
in a becker and permitted to stand there until cooling. After this procedure,
the extract was filtered in paper and the initial volume restored with distilled
water. Decoction: 10 g dried leaves and 100 mL water were added
together in a Becker for boiling for 60 and 90 minutes. After boiling, extract
was filtered in paper and the initial volume replenished with distilled water.
Sterilization: Infusions, aqueous extracts and ethanol extracts
were sterilized through exposition to germicidal (254 nm) ultraviolet
light (UV) at a dose rate of 2 Joules/m2/s for 15
minutes (1800 J/m2), in addition the traditional
steam-sterilization process.

The infusion (900 mL) was partitioned with hexane and ethyl
acetate successively. The aqueous residue was lyophilized and the resulting
solid was solubilized in methanol:water (1:1) to be fractionated by
column chromatography (50 x 2 cm) on XAD-2 (Sigma). Elution
with water yielded 7 fractions named F1, F2, F3, F4, F5, F6 and F7, of which the
first four were considered of good yield. The ethanol extract was partitioned
between several solvents and water. One of the partitions, the ethyl acetate
one, was chromatographed on Sephadex LH-20 (Pharmacia) column (60
x 4 cm) and eluted with methanol:water 1:1 (47 fractions). That
fraction containing flavonoids (named FF) was chromatographed on
Sephadex LH-20 (Pharmacia) column (50 x 2.5 cm) under the
same conditions (39 fractions) above, until complete purification of
the four compounds. These pure compounds were developed after spraying the TLC
chromatogram (stationary phase: silica gel; mobile phase: ethyl
acetate/methanol/ water/acetic acid 80:10:5:5 v/v) with
NP/PEG, a characteristic reagent for flavonoid identification: isovitexin
(Rf value of 0.80), isoorientin (Rf value of 0.50),
swertisin (Rf value of 0.90) and swertiajaponin (Rf value of
0.65). Thus, the compounds were analyzed by 1H,
13C-NMR, HMQC and HMBC techniques and the data were compared with
literature (Cheng et al. 2000, Kumarasamy
et al. 2004).

The thin layer chromatography on silica gel 60 F254 (Merck)
with authentic standards (Sigma-Aldrich) was run for identification of
aminoacids and sugars. Pure aminoacids were run together with samples, with a
solvent system consisting of: ethyl acetate : water : ethanol : acetic acid
(9:2:2:2) and ninhydrin as the developing spraying reagent. Sugars
were chromatographed with a solvent system consisting of: butanol : water :
ethanol : acetic acid (4:1:1:0.5) and orcinol/sulphuric acid as
the developing spraying reagent.

Bacterial Media

Bacterial cells were grown overnight in a shaking incubator at
37°C, in LB medium (Miller
1992). A start inoculum of the lysogenic strain was taken from this
culture and cells were grown in the same medium until their exponential phase
(108 cells/mL). E. coli bacterial
survival and induced infective centers (see below) were scored by
plating samples in LB medium and LB medium containing 20 ug/ mL ampicilin
(LB-amp), respectively. Both media were solidified with 1.5%
Difco bacto agar.

Lysogenic Induction Assay

The E. coli B/r strains
WP2s(λ)(WP2 uvrA (λ)
trpE) and RJF013 (B/r SR714
uvrD3 trpE AmpR) were used
in the lysogenic induction assays, and the protocol was similar to the
quantitative Inductest developed by Moreau et
al. (1976). Each experimental determination was performed
in duplicate, and the results represent the average of at least three
experiments. Test preparations (150 uL) were incubated at 37°C
for 20 min, in the dark, with 100 uL of lysogenic culture, diluted to
104 cells/mL. Then, 0.3 ml of an overnight culture of RJF013
strain and 0.25 mL molten soft agar were added and the mixture was poured onto
LB-amp plates. Plates were incubated overnight at 37°C, and plaques were
scored afterwards. The lysogenic induction was determined as the number of
infective centers per 104 cells. As a negative control, 150 uL pure
water was added to the cultures to measure the spontaneous induction that
averaged 6.8 ± 2.8 infective centers per 104 lysogenic cells. As
a positive control a single UV-C radiation dose (2
J/m2) was given to cultures and the induced infective
centers were scored, with an average 645.8 ± 65.4 infective centers per
104 lysogenic cells. Bacterial survival ranged between 90 and
100% under these conditions.

Sodium Bisulfite Addition and Spectrophotometry

Four systems (A1, B1, A2, B2) were prepared by mixing 20 mL
of E. grandiflorus infusions in 50 ml hydrolysis tubes with
addition of 20 mg sodium bisulfite only in tubes B1 and B2. The tubes were
placed in a boiling water bath for 60 minutes (A1 e B1) and 90 minutes
(A2 e B2). After heating, the tubes were centrifuged at 10000 rpm for
8 minutes (Fogliano et al.
1999). Pellets were discarded and UV-visible spectra of the
supernatants were recorded.

Statistical Analyses

The results are given as average ± standard deviation. Multiple
comparisons were made by analysis of variance (ANOVA).

RESULTS AND DISCUSSION

Echinodorus grandiflorus preparations were tested for
genotoxic activity by means of Escherichia coli lysogenic induction
(Inductest) assay. Prophage-induced bacterial lysis ensues whenever a
genotoxic agent targets the bacterial genome. E. grandiflorus
extracts prepared under different forms were subjected to the Inductest protocol.
Two procedures of sample sterilization were used, steam and UV sterilization. Only
the extract prepared by infusion and steam sterilization resulted positive in the
Inductest assay (Table I).

TABLE I Bacterial Inductest results after exposure to Echinodorus
grandiflorus extracts prepared under different
conditions.

(1)Statistically different means
(p-value<0.0001) (two-way
ANOVA, followed by Bonferroni posttests).

(2)microliter.

(3)SS – steam sterilization – 120°C for 20
minutes.

(4)UVS – ultraviolet sterilization (2
J/m2/s).

Additionaly, increased browning and sweetened smell were observed to
develop in this steam sterilized extract. In search for the compounds responsible
for the observed effects, genotoxic activity of the major fractions (F1, F2, F3
and F4) was evaluated by inductest protocol (Table II).

The genotoxic activity remained in the more polar fractions. The ethyl
acetate partition was not active in the Inductest at the assayed concentration
(5 mg/plate). However, the four flavonoids identified
(isovitexin, isoorientin, swertisin and swertiajaponin) alone were
genotoxic at the tested concentration (5 mg/plate) (Figure 1). Although fraction FF had not
been active in the Inductest, flavonoids were its main constituents. As this class
of compounds can be mutagenic, as described by several researchers, they had to be
purified in order to verify their genotoxicity.

Figure 1 - Synthesis of principal results.

Flavonoids are well known antioxidant, anticarcinogenic and antimutagenic
natural compounds. However, if present in higher concentrations, these substances
can be pro-oxidants and elicit mutagenic responses (Rietjens et al. 2005). Noteworthy is that the identified
C-glycosylflavonoids are well characterized antioxidants and xanthine oxidase
inhibitors (Pham et al. 2013). This
can explain the popular use of the plant for treatment of diseases of the
genitourinary tract.

During the chemical fractionation of steam sterilized infused extract, an
aromatic smell and browning aspect developed, and compounds appeared to remain in
fractions F1 and F2 (Figure 1). A
Maillard's (Maillard 1912)
reaction was then envisaged to explain the observed genotoxic effects and further
investigated. First of all, the presence of reducing sugars and aminoacid precursors
in the infusion was investigated by TLC. Aspartic acid, threonine, serine, valine,
methionine, alanine, glutamic acid, phenylalanine, proline, arginine, tyrosine,
leucine, glycine, lysine and cysteine were the identified aminoacids and galactose,
glucose, maltose and lactose, the reducing sugars. Thus, control solutions
A, 10% glucose, B, 10% glucose plus
0.5% tryptophan, and C, 10% glucose plus an aminoacid mixture
(according to description provided in Materials and Methods) were prepared
and steam sterilized. As expected, aromatic smell and browning developed in
solutions B and C (absorbance at 420 nm, see Table III).

TABLE III Smell releasing, browning and absorbance inspection as evidence for
development of Maillard's Reaction.

(1)unpaired Student's t-test comparing absorbance
mean before and after stem sterilization for each solution.

Changes in organoleptic properties of solutions B and C were suggestive
of the presence of Maillard's products. The Maillard's characteristic
browning color is due to melanoidin formation in heated preparations. Recently,
products of Maillard's reaction have been related to enhanced mutagenicity,
connected with increased accumulation of reactive oxygen species and DNA damage
(Janzowski et al. 2000, Monnier 2003, Coca et al. 2004, Kwak et al.
2005). Literature surveys (Friedman 1996, Namiki 1988)
about browning appearance in preparations subjected to heating processes indicated
that it develops proportionally to aminoacid concentration and reduced capacity of
the reactant sugars. Martins and Van Boekel (2003) have suggested a random
polymerization of heat-degradation products of sugars with amino groups, like those
present in aminoacids. The precise mechanism of melanoidin formation (brown
color) and its structure are not yet fully understood. Time, temperature, pH
and water quantity are determinant factors influencing the extent of the reaction
(Martins and Van Boekel 2003, Van Boekel 2006).

Both steam sterilized infusions and UV sterilized decoctions of
Echinodorus grandiflorus were assayed by the Inductest
protocol. Lysogenic induction was shown to increase after exposure of test bacterial
cultures to decocts when compared to that observed for infusions preparations
(Table IV).

Lysogenic induction was seen to increase only when samples were steam
sterilized (120°C, 20 min). However, the decoction - an extractive
technique involving longer boiling times and contact among reactant compounds than
infusion-prepared ones - caused browning formation (as measured by absorption
at 420 nm) and genotoxicity. Melanoidin formation depends on strict reaction
kinetics. It appears steeply with increasing times of decoction (Cuzzoni et al. 1988). Nevertheless, Baisier and Labuza (1992) while
analyzing melanoidin formation by fluorescence spectroscopy found decay in
melanoidin fluorescence whenever longer times were given for Maillard's
reaction to occur. They suggested that degradation of melanoidin pigment could
somehow explain the phenomenon. Accordingly, decoction for 60 min caused more
melanoidin to form and lysogenic activity than that carried out for 90 min in this
study.

Finally, Maillard's reaction was partially inhibited when decoction
of Echinodorus grandiflorus leaves was prepared in the presence of
0.1% sodium bisulfite, a well-known inhibitor for that reaction. Sodium
bisulfite caused 420 nm absorbance to drop in decocts, in comparison with control
samples. In Table IV, a drop in 420 nm
absorbance (0.4291) can be seen when preparations were boiled for 60 min
in comparison with the absorbance value of 0.4954 found for decoction during 60 min
without sodium bisulfite addition. The initial 420 nm absorbance of
Echinodorus grandiflorus' infusion was 0.3774.

Maillard's-induced melanoidins pigments responsible for browning
color in foods are polymeric compounds of pyrrol and furan rings formed during more
advanced stages of the reaction (Tressl et al.
1998). Remarkably, this is the first paper reporting in the
appearance of such compounds in a phytomedicine. Mutagenic and genotoxic activities
of those compounds were widely described (Kim
et al. 1991, Powrie et al. 1981,
Monnier 2003, Jägerstad and Skog 2005). Attention has to be paid if
these compounds appear in phytomedicines.

CONCLUSION

Steam sterilization of infusion and decoction preparations of
Echinodorus grandiflorus led to formation of Maillard's
reaction products, as seen by the browning formation and the releasing of
characteristic smell. Bio-guided fractionation by Inductest led to isolation of the
flavonoids isovitexin, isoorientin, swertisin and swertiajaponin, which, in turn,
were devoid of genotoxic effects when tested separately. Since Maillard's
reaction precursors were casted in such aqueous fractions, i.e.,
aminoacids and reducing sugars, we have concluded that melanoidins, well-known
mutagenic and genotoxic compounds can be formed in such preparations. The
phythomedicines and other derived formulations need to be assessed in relation to
melanoidin formation, as is widely verified in the food industry, to assure safeness
for human consumption.

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